U.S. patent application number 10/564340 was filed with the patent office on 2006-09-21 for conjugated biological molecules and their preparation.
Invention is credited to Stephen James Brocchini, Jin-Won Choi, Antony Robert Godwin, Elisa Pedone, Sunil Shaunak.
Application Number | 20060210526 10/564340 |
Document ID | / |
Family ID | 27742023 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060210526 |
Kind Code |
A1 |
Brocchini; Stephen James ;
et al. |
September 21, 2006 |
Conjugated biological molecules and their preparation
Abstract
Novel biologically active compounds of the general formula (I)
in which one of X and X' represents a polymer, and the other
represents a hydrogen atom; each Q independently represents a
linking group; W represents an electron-withdrawing moiety or a
moiety preparable by reduction of an electron-withdrawing moiety;
or, if X' represents a polymer, X-Q-W- together may represent an
electron withdrawing group; and in addition, if X represents a
polymer, X' and electron withdrawing group W together with the
interjacent atoms may form a ring; each of Z.sup.1 and Z.sup.2
independently represents a group derived from a biological
molecule, each of which is linked to A and B via a nucleophilic
moiety; or Z.sup.1 and Z.sup.2 together represent a single group
derived from a biological molecule which is linked to A and B via
two nucleophilic moieties; A is a C.sub.1-5 alkylene or alkenylene
chain; and B is a bond or a C.sub.1-4 alkylene or alkenylene chain;
are formed by conjugating a suitable polymer to a suitable
biologically active molecule via nucleophilic groups in said
molecule, preferably via a disulphide bridge. ##STR1##
Inventors: |
Brocchini; Stephen James;
(Welwyn Garden, GB) ; Godwin; Antony Robert;
(Bristol, GB) ; Pedone; Elisa; (Leytonstone,
GB) ; Choi; Jin-Won; (Leytonstone, GB) ;
Shaunak; Sunil; (Hertfordshire, GB) |
Correspondence
Address: |
BELL & ASSOCIATES
416 FUNSTON ST., SUITE 100
SAN FRANCISCO
CA
94118
US
|
Family ID: |
27742023 |
Appl. No.: |
10/564340 |
Filed: |
July 12, 2004 |
PCT Filed: |
July 12, 2004 |
PCT NO: |
PCT/GB04/02994 |
371 Date: |
January 11, 2006 |
Current U.S.
Class: |
424/78.27 ;
525/54.1; 530/395 |
Current CPC
Class: |
A61P 37/02 20180101;
A61K 47/60 20170801 |
Class at
Publication: |
424/078.27 ;
530/395; 525/054.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 31/785 20060101 A61K031/785; C08G 63/91 20060101
C08G063/91; C08G 63/48 20060101 C08G063/48 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 11, 2003 |
GB |
0316294.8 |
Claims
1-14. (canceled)
15. A process for preparing a compound of the general formula
##STR17## in which one of X and X' represents a polymer, and the
other represents a hydrogen atom; each Q independently represents a
linking group; W represents an electron-withdrawing moiety or a
moiety preparable by reduction of an electron-withdrawing moiety;
or, if X' represents a polymer, X-Q-W- together may represent an
electron withdrawing group; and in addition, if X represents a
polymer, X' and electron withdrawing group W together with the
interjacent atoms may form a ring; each of Z.sup.1 and Z.sup.2
independently represents a group derived from a biological
molecule, each of which is linked to A and B via a nucleophilic
moiety; or Z.sup.1 and Z.sup.2 together represent a single group
derived from a biological molecule which is linked to A and B via
two nucleophilic moieties; A is a C.sub.1-5 alkylene or alkenylene
chain; and B is a bond or a C.sub.1-4 alkylene or alkenylene chain;
wherein the process comprises reacting either (i) a compound of the
general formula ##STR18## in which X, X', Q, A and B have the
meanings given for the general formula I; W' represents an
electron-withdrawing group or, if X' represents a polymer, X-Q-W'
together may represent an electron withdrawing group; and each L
independently represents a leaving group; or (ii) a compound of the
general formula ##STR19## in which X, X', Q, W', A and L have the
meanings given for the general formula II, and in addition if X
represents a polymer, X' and electron-withdrawing group W' together
with the interjacent atoms may form a ring, and m represents an
integer 1 to 4; with compounds of the general formula Z.sup.1 Nu or
Z.sup.2Nu in which each of Z.sup.1 and Z.sup.2 independently
represents a group derived from a biological molecule, or a
compound of the formula Z(Nu).sub.2 in which Z represents a
biological molecule, and each Nu independently represents a
nucleophilic group; and optionally converting a resulting compound
of the formula I in which W is an electron-withdrawing group into a
corresponding compound of the formula (I) by reduction of the group
W'.
16. The process as claimed in claim 15, in which a polymer X or X'
is a homo- or copolymer selected from the group consisting of
polyalkylene glycols, polyvinylpyrrolidones, polyacrylates,
polymethacrylates, polyoxazolines, polyvinylalcohols,
polyacrylamides, polymethacrylamides, HPMA copolymers, polyesters,
polyacetals, poly(ortho ester)s, polycarbonates, poly(imino
carbonate)s, polyamides, copolymers of divinylether-maleic
anhydride or styrene-maleic anhydride, polysaccharides, or
polyglutamic acids, any of said homo- or co-polymers optionally
being derivatized or functionalized.
17. The process as claimed in claim 16, in which the polymer is a
polyethylene glycol.
18. The process as claimed in claim 15, in which each linking group
Q independently represents a direct bond, an alkylene group, or an
optionally-substituted aryl or heteroaryl group, any of which may
be terminated or interrupted by one or more oxygen atoms, sulphur
atoms, --NR groups in which R represents an alkyl or aryl group,
keto groups, --O--CO-- groups and/or --CO--O-- groups.
19. The process as claimed in claim 15, in which W' represents a
keto or aldehyde group CO, an ester group --O--CO-- or a sulphone
group --SO.sub.2--.
20. The process as claimed in claim 15, in which the compound of
formula (II) or (III) is reacted with a compound of the formula
Z(Nu).sub.2 in which Z represents a biological molecule.
21. The process as claimed in claim 20, in which Z represents a
protein.
22. The process as claimed in claim 15, in which each of Z.sup.1
and Z.sup.2 becomes linked to A and B via thiol groups.
23. The process as claimed in claim 21, in which Z becomes linked
to A and B via thiol groups.
24. The process as claimed in claim 15, in which the or each
leaving group L represents --SR, --SO.sub.2R, --OSO.sub.2R,
--N.sup.+R.sub.3, --N.sup.+HR.sub.2, --N.sup.+H.sub.2R, halogen, or
--OO, in which R represents an alkyl or aryl group and O represents
a substituted aryl group containing at least one electron
withdrawing substituent.
25. A compound comprising the general formula II ##STR20## in which
one of X and X' represents a polymer, and the other represents a
hydrogen atom; each Q independently represents a linking group; W'
represents an electron-withdrawing group or, if X' represents a
polymer, X-Q-W' together may represent an electron withdrawing
group; A is a C.sub.1-5 alkylene or alkenylene chain; and B is a
bond or a C.sub.1-4 alkylene or alkenylene chain; and each L
independently represents a leaving group.
26. A compound having the general formula III ##STR21## in which
one of X and X' represents a polymer, and the other represents a
hydrogen atom; each Q independently represents a linking group; W'
represents an electron-withdrawing group or, if X' represents a
polymer, X-Q-W' together may represent an electron withdrawing
group; and in addition if X represents a polymer, X' and
electron-withdrawing group W' together with the interjacent atoms
may form a ring; A is a C.sub.1-5 alkylene or alkenylene chain; B
is a bond or a C.sub.1-4 alkylene or alkenylene chain; each L
independently represents a leaving group; and m represents an
integer 1 to 4.
27. A compound having the general formula I ##STR22## in which one
of X and X' represents a homo- or copolymer selected from the group
consisting of polyalkylene glycols, polyvinylpyrrolidones,
polyacrylates, polymethacrylates, polyoxazolines,
polyvinylalcohols, polyacrylamides, polymethacrylamides, HPMA
copolymers, polyesters, polyacetals, poly(ortho ester)s,
polycarbonates, poly(imino carbonate)s, copolymers of
divinylether-maleic anhydride or styrene-maleic anhydride,
polysaccharides, or polyglutamic acids, any of said homo- or
co-polymers optionally being derivatized or functionalized; and the
other represents a hydrogen atom; each Q independently represents a
linking group; W represents an electron-withdrawing moiety or a
moiety preparable by reduction of an electron-withdrawing moiety;
or, if X' represents a polymer, X-Q-W- together may represent an
electron withdrawing group; and in addition, if X represents a
polymer, X' and electron withdrawing group W together with the
interjacent atoms may form a ring; each of Z.sup.1 and Z.sup.2
independently represents a group derived from a biological
molecule, each of which is linked to A and B via a nucleophilic
moiety; or Z.sup.1 and Z.sup.2 together represent a single group
derived from a biological molecule which is linked to A and B via
two nucleophilic moieties; A is a C.sub.1-5 alkylene or alkenylene
chain; and B is a bond or a C.sub.1-4 alkylene or alkenylene
chain.
28. The compound as claimed in claim 27, in which the polymer X or
X' is a polyethylene glycol.
29. The compound as claimed in claim 27, in which each linking
group Q independently represents a direct bond, an alkylene group,
or an optionally-substituted aryl or heteroaryl group, any of which
may be terminated or interrupted by one or more oxygen atoms,
sulphur atoms, --NR groups in which R represents an alkyl or aryl
group, keto groups, --O--CO-- groups and/or --CO--O-- groups.
30. The compound as claimed in claim 27, in which W represents a
keto or aldehyde group CO, an ester group --O--CO-- or a sulphone
group --SO.sub.2--, or a group obtained by reduction of such a
group, or X-Q-W- together represent a cyano group.
31. The compound as claimed in claim 27, in which Z.sup.1 and
Z.sup.2 together represent a single biological molecule.
32. The compound as claimed in claim 31, in which Z.sup.1 and
Z.sup.2 together represent a protein.
33. The compound as claimed in claim 32, in which the protein is
linked to A and B via thiol groups.
34. The compound as claimed in claim 33, in which said thiol groups
have been generated by partial reduction of a disulphide
bridge.
35. A pharmaceutical composition comprising a physiologically
tolerable compound as claimed in claim 27, together with a
pharmaceutically acceptable carrier.
36. A method for treating a patient, the method comprising
administering a pharmaceutically-effective amount of the
pharmaceutical composition as claimed in claim 35 to the patient.
Description
[0001] This invention relates to conjugated biological molecules
and their preparation from novel chemically functionalised
derivatives of polymers such as polyethylene glycol.
[0002] Many therapeutically active molecules do not possess the
properties required to achieve efficacy in clinical medical use.
For example, therapeutically active proteins and poly-peptides are
now being discovered and produced by the biopharmaceutical industry
and by genetic engineering. Although there are currently at least
80 protein based medicines marketed in the United States with at
least 350 more protein based medicines undergoing clinical trails
(Harris J, Chess R: Effect of Pegylation on pharmaceuticals. Nature
Review Drug Discovery, 2003, 2, 214-221), most native proteins do
not make good medicines because upon administration to patients
there are several inherent drawbacks that include: (1) proteins are
digested by many endo-and exopeptidases present in blood or tissue,
(2) many proteins are immunogenic to some extent and (3) proteins
can be rapidly excreted by kidney ultrafiltration. Other molecules
used as active therapeutic agents in medicines that are
systemically toxic or lack optimal bioavailability and
pharmacokinetics include low molecular weight molecules where an
effective dose is limited by toxicity. Such molecules are routinely
used to treat inflammation and conditions due to malignancies,
infection and autoimmune disease.
[0003] Water soluble, synthetic polymers, particularly polyalkylene
glycols, are used to conjugate therapeutically active molecules
such as proteins. These therapeutic conjugates have been shown to
favourably alter pharmacokinetics by prolonging circulation time
and decreasing clearance rates, decrease systemic toxicity, and in
several cases, to display increased clinical efficacy. This process
of covalently conjugating polyethylene glycol, PEG, to proteins is
commonly known as "PEGylation" however many different polymers have
been examined as conjugating polymers.
[0004] Many polymer reagents for conjugation comprise conjugating
chemical functionality that is hydrolytically unstable. Examples of
hydrolytically unstable polymeric conjugating reagents are active
esters that include, for example, polyalkylene oxide-N-succinimide
carbonates (Zalipsky U.S. Pat. No. 5,122,614). These reagents have
relatively short half lives in aqueous media, that includes blood
or plasma. This results in the need to add large stoichiometric
excesses of the conjugating polymer reagent. The hydrolytic
stability of the reagent is important because the requirement to
add stoichiometric excesses for protein conjugation requires
significant effort and cost to purify the polymer-protein conjugate
from the reaction mixture. Furthermore, these hydrolytically
unstable reagents tend to undergo preferentially, reaction with
amine chemical functionality in the protein, particularly to the
e-amine of lysine residues. Since most proteins of interest have
more than one lysine residue, and frequently many lysine residues,
then conjugation tends to be non-specific in that it occurs at many
residue sites on the protein. It is possible to purify the
conjugating reaction mixture to isolate proteins conjugated to one
polymer molecule, however it is not possible, to isolate at a
reasonable cost, polymer-protein conjugates that are all conjugated
to the same amine group on the protein. Non-specific conjugation
frequently results in impaired protein function. For example
antibodies and antibody fragments with random poly(alkylene oxide)
attachment via lysine residues result in modified antibodies (or
modified antibody fragments) able to bind target antigen with
reduced affinity, avidity or specificity. Additionally, amine
specific polymer conjugating reagents require conjugating reaction
conditions that must be selected to ensure that the amines on the
protein are not protonated. These conditions require moderately
high pH media (8-10), this allows the amine moieties to be reactive
enough for reaction with the polymer conjugating reagent. High pH
conditions are frequently deleterious to the protein causing
structural changes and denaturation. These processes result in
impairment of protein function. Amine specific polymer conjugation
reagents tend to bind to accessible amine sites on the protein.
These reagents can be termed kinetic reagents. They are labile and
undergo a reaction with the most assessable amino nucleophilic
sites on the protein. Amine specific polymer conjugating reagents
that conjugate by amine acylation result in the loss of positive
charge on the amine group of the amino acid residue on the protein
that would normally be present under physiological conditions for
the unconjugated protein. These features of amine specific polymer
conjugating reagents often leads to partial impairment of the
function of the protein. Other conjugating functional groups
incorporated in polymers for conjugation to protein and that are
amine specific and frequently hydrolytically labile include
isocyanate (WO 94/04193) and carbonates (WO 90/13540).
[0005] Particularly relevant for optimised efficacy and to ensure
dose to dose consistency is to make certain that the number of
conjugated polymer molecules per protein is the same and that each
polymer molecule is specifically covalently conjugated to the same
amino acid residue in each protein molecule. Non-specific
conjugation at sites along a protein molecule results in a
distribution of conjugation products and frequently, unconjugated
protein, to give a complex mixture that is difficult, tedious, and
expensive to purify.
[0006] Thiol specific polymer conjugating reagents for proteins
have been developed to address the limitations for the propensity
of the conjugating reagent to undergo hydrolysis that is
competitive with conjugation to the protein, non-specific polymer
conjugation at different amino acid residues in the protein, and
the need for high pH conjugating reaction conditions. Thiol
specific polymer conjugating reagents can be utilised at pH values
close to neutrality where the amine functional moieties on the
amino acid residues of the protein are protonated and thus cannot
effectively compete in the conjugation reaction with the polymer
conjugating reagent. Thiol specific polymer conjugating reagents
that are relatively more hydrolytically stable than are the
aforementioned amine specific reagents can be utilised at a lower
stoichiometric excess thus reducing the cost during purification of
the polymer-protein conjugate. Conjugating functional moieties that
are broadly selective for thiol groups include iodoacetamide,
maleiimide (WO 92/16221), vinylsulfone (WO 95/13312 and WO
95/34326), vinyl pyridines (WO 88/05433), and acrylate and
methacrylate esters (WO 99/01469). These thiol selective
conjugating moieties yield a single thioether conjugating bond
between the polymer.
[0007] Most proteins do not have free sulfhydrals because these
sulfhydrals undergo rearrangement and scrambling reactions with the
disulfide bridges within the protein resulting in impaired protein
function. For proteins that do have free sulfhydrals, these
sulfhydrals are frequently critical for protein function. Typically
in a protein, the number of sulfhydral moieties is less than the
number of amine moieties (e.g. lysine or histadine). Since
conjugation to a protein can be made to be specific at thiol groups
and since proteins do not typically have free thiol groups, there
are examples of site-specific modification of protein by
mutagenesis to introduce thiol sites for PEG attachment. However
such modifications increase costs significantly. The introduced
free sulhydral can have the similar limitations as mentioned
heretofore in the engineered protein for protein scrambling and
protein dimerisation. Also the process of mutagensis and production
of the modified protein from bacterial sources frequently causes
the free sulfhydral to be bound in a disulfide bond with
glutathione, for example. Interleukin-2, for example, has been
modified by mutagenesis to replace a threonine residue by a
cysteine to allow site specific attachment of PEG. [Goodson R J,
Katre N V; Bio/Technology (1990) 8, 343-346].
[0008] It is known in the art that conjugating parameters have to
be optimally matched with the therapeutically active molecule of
interest in terms of polymer morphology, molecular weight
characteristics, chemical functionality. Although the polymer
protein conjugate, can display many favourable and necessary
properties needed for safe, effective medical use, the effect of
polymer conjugation on the activity and stability of the protein is
of vital importance for performance. Conjugation variables related
to the location and amount of conjugation and polymer
characteristics must be optimally correlated with biological and
physicochemical properties.
[0009] We have now found a series of novel reagents which can be
used inter alia to conjugate with both sulphur atoms derived from
two cysteine residues in a protein to give novel thioether
conjugates. The invention in the first instance is intended for the
conjugation of the two sulphur atoms that form natural disulphide
bridges in native proteins. Disulphide bonds are found in medically
relevant proteins, specifically, secretory proteins, lysosomal
proteins, and the exoplasmic domains of membrane proteins. The
technology provides clear advantages over known techniques for
conjugating polymers to proteins.
[0010] The present invention provides a compound of the general
formula ##STR2## [0011] in which one of X and X' represents a
polymer, and the other represents a hydrogen atom; [0012] each Q
independently represents a linking group; [0013] W represents an
electron-withdrawing moiety or a moiety preparable by reduction of
an electron-withdrawing moiety; or, if X' represents a polymer,
X-Q-W- together may represent an electron withdrawing group; and in
addition, if X represents a polymer, X' and electron withdrawing
group W together with the interjacent atoms may form a ring; [0014]
each of Z.sup.1 and Z.sup.2 independently represents a group
derived from a biological molecule, each of which is linked to A
and B via a nucleophilic moiety; or Z.sup.1 and Z.sup.2 together
represent a single group derived from a biological molecule which
is linked to A and B via two nucleophilic moieties; [0015] A is a
C.sub.1-5 alkylene or alkenylene chain; and [0016] B is a bond or a
C.sub.1-4 alkylene or alkenylene chain.
[0017] A polymer X or X' may for example be a polyalkylene glycol,
a polyvinylpyrrolidone, a polyacrylate, for example polyacryloyl
morpholine, a polyoxazoline, a polyvinylalcohol, a polyacrylamide
or polymethacrylamide, for example polycarboxymethacrylamide, or a
HPMA copolymer. Additionally X or X' may be a polymer that is
susceptible to enzymatic or hydrolytic degradation. Such polymers,
for example, include polyesters, polyacetals, poly(ortho esters),
polycarbonates, poly(imino carbonates), and polyamides, such as
poly(amino acids). A polymer X or X' may be a homopolymer, random
copolymer or a structurally defined copolymer such as a block
copolymer. For example X or X' may be a block copolymer derived
from two or more alkylene oxides, or from poly(alkylene oxide) and
either a polyester, polyacetal, poly(ortho ester), or a poly(amino
acid). Polyfunctional polymers that may be used include copolymers
of divinylether-maleic anhydride and styrene-maleic anhydride.
Naturally occurring polymers may also be used, for example
polysaccharides such as chitin, dextran, dextrin, chitosan, starch,
cellulose, glycogen, poly(sialylic adic) and derivatives thereof.
Polymers such as polyglutamic acid may also be used, as may hybrid
polymers derived from natural monomers such as saccharides or amino
acids and synthetic monomers such as ethylene oxide or methacrylic
acid.
[0018] If the polymer is a polyalkylene glycol, this is preferably
one containing C.sub.2 and/or C.sub.3 units, and is especially a
polyethylene glycol. A polymer, particularly a polyalkylene glycol,
may contain a single linear chain, or it may have branched
morphology composed of many chains either small or large. The
so-called Pluronics are an important class of PEG block copolymers.
These are derived from ethylene oxide and propylene oxide blocks.
Substituted polyalkylene glycols, for example methoxypolyethylene
glycol, may be used. In a preferred embodiment of the invention, a
single-chain polyethylene glycol is initiated by a suitable group,
for example an alkoxy, e.g. methoxy, aryloxy, carboxy or hydroxyl
group, and is connected at the other end of the chain to the linker
group Q.
[0019] The polymer X or X' may optionally be derivatised or
functionalised in any desired way. For example, polymers with two
or more chemical moieties for conjugation that are the subject of
this invention may be used to create conjugates of two or more
linked bioactive molecules. Reactive groups may be linked at the
polymer terminus or end group, or along the polymer chain through
pendent linkers; in such case, the polymer is for example a
polyacrylamide, polymethacrylamide, polyacrylate, polymethacrylate,
or a maleic anhydride copolymer. Multimeric conjugates that contain
more than one biological molecule, typically a biologically active
polypeptide or drug can result in synergistic and additive
benefits. If desired, the polymer may be coupled to a solid support
using conventional methods.
[0020] The optimum molecular weight of the polymer will of course
depend upon the intended application. Preferably, the number
average molecular weight is in the range of from 500 g/mole to
around 75,000 g/mole. When the compound of the general formula I is
intended to leave the circulation and penetrate tissue, for example
for use in the treatment of inflammation caused by malignancy,
infection or autoimmune disease, or by trauma, it may be
advantageous to use a lower molecular weight polymer in the range
2000-30,000 g/mole. For applications where the compound of the
general formula I is intended to remain in circulation it may be
advantageous to use a higher molecular weight polymer, for example
in the range of 20,000-75,000g/mole.
[0021] The polymer to be used should be selected so the conjugate
is soluble in the solvent medium for its intended use. For
biological applications, particularly for diagnostic applications
and therapeutic applications for clinical therapeutical
administration to a mammal, the conjugate will be soluble in
aqueous media. Many proteins such as enzymes have utility in
industry, for example to catalyze chemical reactions. For
conjugates intended for use in such applications, it may be
necessary that the conjugate be soluble in either or both aqueous
and organic media. The polymer should not impair the intended
function of the biological molecule(s).
[0022] A linking group Q may for example be a direct bond, an
alkylene group (preferably a C.sub.1-10 alkylene group), or an
optionally-substituted aryl or heteroaryl group, any of which may
be terminated or interrupted by one or more oxygen atoms, sulphur
atoms, --NR groups (in which R has the meaning given below), keto
groups, --O--CO-- groups and/or --CO--O-- groups. Suitable aryl
groups include phenyl and naphthyl groups, while suitable
heteroaryl groups include pyridine, pyrrole, furan, pyran,
imidazole, pyrazole, oxazole, pyridazine, primidine and purine. The
linkage to the polymer may be by way of a hydrolytically labile
bond, or by a non-labile bond.
[0023] Substituents which may be present on an optionally
substituted aryl or heteroaryl group include for example one or
more of the same or different substituents selected from --CN,
--NO.sub.2, --CO.sub.2R, --COH, --CH.sub.2OH, --COR, --OR, --OCOR,
--OCO.sub.2R, --SR, --SOR, --SO.sub.2R, --NHCOR, --NRCOR,
--NHCO.sub.2R, --NR'CO.sub.2R, --NO, --NHOH, --NR'OH,
--C.dbd.N--NHCOR, --C.dbd.N--NR'COR, --N.sup.+R.sub.3,
--N.sup.+H.sub.3, --N.sup.+HR.sub.2, --N.sup.+H.sub.2R, halogen,
for example fluorine or chlorine, --C.ident.CR, --C.dbd.CR.sub.2
and 13 C.dbd.CHR, in which each R or R' independently represents a
hydrogen atom or an alkyl (preferably C.sub.1-6 alkyl) or an aryl
(preferably phenyl) group. The presence of electron withdrawing
substituents is especially preferred.
[0024] W may for example represent a keto or aldehyde group CO, an
ester group --O--CO-- or a sulphone group --SO.sub.2--, or a group
obtained by reduction of such a group, e.g. a CH.OH group, an ether
group CH.OR, an ester group CH.O.C(O)R, an amine group CH.NH.sub.2,
CH.NHR or CH.NR.sub.2, or an amide CH.NHC(O)R or CH.N(C(O)R).sub.2.
If X-Q-W- together represent an electron withdrawing group, this
group may for example be a cyano group.
[0025] In the following portion of this specification, Z.sup.1 and
Z.sup.2 will be referred to collectively as Z. It is a preferred
embodiment of the invention that Z.sup.1 and Z.sup.2 together
should represent a single biological molecule: ##STR3##
[0026] Z may be derived from any desired biological molecule, for
example a protein. The protein may for example be a polypeptide,
antibody, antibody fragment, enzyme, cytokine, chemokine or
receptor. Constrained or cyclic polypeptides, which are usually
cyclised through a disulphide bridge, and epitopes, may also be
used. Specific examples of suitable biological molecules are listed
below.
[0027] Preferably a nucleophilic moiety linking A or B to the
group(s) Z is derived from a thiol group or amine group. Two thiol
groups may be generated by partial reduction of a natural or
engineered disulphide (cysteine) bridge. Amine groups may for
example be lysine residues. Where Z.sup.1 and Z.sup.2 together form
a single biological molecule which is linked to the groups A and B
via two thiol groups, the compound of the formula I has the formula
##STR4##
[0028] The invention also provides a process for the preparation of
a compound of the general formula I, which comprises reacting
either (i) a compound of the general formula ##STR5## in which one
of X and X' represents a polymer and the other represents a
hydrogen atom; [0029] Q represents a linking group; [0030] W'
represents an electron-withdrawing group, for example a keto group,
an ester group --O--CO-- or a sulphone group --SO.sub.2--; or, if
X' represents a polymer, X-Q-W' together may represent an electron
withdrawing group; [0031] A represents a C.sub.1-5 alkylene or
alkenylene chain; [0032] B represents a bond or a C.sub.1-4
alkylene or alkenylene chain; and [0033] each L independently
represents a leaving group; or (ii) a compound of the general
formula ##STR6## in which X, X', Q, W', A and L have the meanings
given for the general formula II, and in addition if X represents a
polymer, X' and electron-withdrawing group W' together with the
interjacent atoms may form a ring, and m represents an integer 1 to
4; with a compound of the general formula Z(Nu).sub.2 (IV) in which
Z has the meaning given above and each Nu independently represents
a nucleophilic group, for example a thiol or an amine group. If
Z.sup.1 and Z.sup.2 are separate molecules, the reaction takes
place in two successive steps with successive molecules Z.sup.1Nu
and Z.sup.2Nu.
[0034] The or each leaving group L may for example represent --SR,
--SO.sub.2R, --OSO.sub.2R, --N.sup.+R.sub.3, --N.sup.+HR.sub.2,
--N.sup.+H.sub.2R, halogen, or --OO, in which R has the meaning
given above, and O represents a substituted aryl, especially
phenyl, group, containing at least one electron withdrawing
substituent, for example --CN, --NO.sub.2, --CO.sub.2R, --COH,
--CH.sub.2OH, --COR, --OR, --OCOR, --OCO.sub.2R, --SR, --SOR,
--SO.sub.2R, --NHCOR, --NRCOR, --NHCO.sub.2R, --NR'CO.sub.2R, --NO,
--NHOH, --NR'OH, --C.dbd.N--NHCOR, --C.dbd.N--NR'COR,
--N.sup.+R.sub.3, --N.sup.+HR.sub.2, --N.sup.+H.sub.2R, halogen,
especially chlorine or, especially, fluorine, --C.ident.CR,
--C.dbd.CR.sub.2 and --C.dbd.CHR, in which R and R' have the
meanings given above.
[0035] Typical structures in which W' and X' together form a ring
include ##STR7## [0036] in which n is an integer from 1 to 4, and
##STR8##
[0037] A compound of the general formula (IV) in which each Nu is a
thiol group may be prepared by partial reduction of a protein
contain a cysteine link, i.e. ##STR9##
[0038] Suitably, the process according to the invention is carried
out by partially reducing a disulfide bond derived from two
cysteine amino acids in the protein in situ following which the
reduced product reacts with the compound of formula (II) or (III).
Disulfides can be reduced, for example, with dithiothreitiol,
mercaptoethanol, or tris-carboxyethylphosphine using conventional
methods. The process may be carried out in a solvent or solvent
mixture in which all reactants are soluble. The biological molecule
containing nucleophilic groups (e.g. protein) may be allowed to
react directly with the compound of the general formula II or III
in an aqueous reaction medium. This reaction medium may also be
buffered, depending on the pH requirements of the nucleophile. The
optimum pH for the reaction is generally between about 5.5 and
about 8, for example about 7.4, preferably about 6.0-6.5. Reaction
temperatures between 3-37.degree. C. are generally suitable:
proteins and other biological molecules may decompose or denature
impairing function if the conjugation reaction is conducted at a
temperature where these processes may occur. Reactions conducted in
organic media (for example THF, ethyl acetate, acetone) are
typically conducted at temperatures up to ambient, for example
temperatures below 0.degree. C.
[0039] A protein can contain one or a multiplicity of disulfide
bridges. Reduction to give free sulfhyral moieties can be conducted
to reduce one or a multiplicity of disulfide bridges in a protein.
Depending on the extent of disulfide reduction and the
stoichiometry of the polymeric conjugation reagent that is used, it
is possible to conjugate one or a multiplicity of polymer molecules
to the protein. Immobilised reducing agents may be used if it is
desired to reduce less than the total number of disulfides, as can
partial reduction using different reaction conditions or the
addition of denaturants.
[0040] Alternatively the source of the thiol groups can be from
cysteines or thiols not originally derived from a disulfide bridge.
If the source of the thiol groups is a disulfide bridge, this may
be intrachanin or interchain.
[0041] The biological molecule can be effectively conjugated with
the reagents of the present invention using a stoichiometric
equivalent or a slight excess of reagent, unlike many prior art
reagents. However, since the reagents of the present invention do
not undergo competitive reactions with aqueous media used to
solvate proteins, it is possible to conduct the conjugation
reaction with an excess stoichiometry of reagent. The excess
reagent can be easily removed by ion exchange chromatography during
routine purification of proteins.
[0042] The compounds of formulae (II) and (III) are novel, and
accordingly the invention further provides these compounds per se.
These novel reagents provide a breakthrough in conjugate
technology, the chemical functional moiety on the polymer
comprising a cross-functionalised, latently cross-conjugated,
bis-alkylating moiety that is selective for two nucleophiles,
particularly two thiols derived from a natural disulfide bond in
proteins.
[0043] The immediate product of the process according to the
invention is a compound of the general formula I in which W is an
electron-withdrawing group. Such compounds have utility in
themselves; because the process of the invention is reversible
under suitable conditions,additionally compounds of formula (I) in
which W is an electron-withdrawing moiety have utility in
applications where release of the free protein is required, for
example in direct clinical applications. An electron-withdrawing
moiety W may, however, be reduced to give a moiety which prevents
release of the protein, and such compounds will also have utility
in many clinical, industrial and diagnostic applications.
[0044] Thus, for example, a moiety W containing a keto group may be
reduced to a moiety W containing a CH(OH) group; an ether group
CH.OR may be obtained by the reaction of a hydroxy group with an
etherifying agent; an ester group CH.O.C(O)R may be obtained by the
reaction of a hydroxy group with an acylating agent; an amine group
CH.NH.sub.2, CH.NHR or CH.NR.sub.2 may be prepared from a ketone or
aldehyde by reductive amination); or an amide CH.NHC(O)R or
CH.N(C(O)R).sub.2 may be formed by acylation of an amine). A group
X-Q-W- which is a cyano group may be reduced to an amine group.
[0045] A compound of the general formula (II) in which X represents
a polymer may be prepared by reacting a compound of the general
formula ##STR10## [0046] in which Q', W, A, B and L have the
meanings given above, with a polymer of the general formula X-V
(VII) in which X represents a polymer; Q' and V being groups
selected such that the compounds of (VI) and (VII) will react
together to give the desired compound of the formula (II).
Alternatively, a compound of the formula ##STR11## may be reacted
with a polymer of the general formula X-Q' (IX)
[0047] A compound of the general formula (III) may be prepared by
base mediated elimination of one leaving group L from a compound of
the general formula (II).
[0048] Compounds of the general formula I may include an imaging
agent, for example a radio nucleotide, to enable tracking of the
compound in vivo. Suitably the radio nucleotide or imaging agent I
may be bound through the group W, to give, for example, compounds
of the type ##STR12## which may for example be prepared from
reagents of the type ##STR13## for example ##STR14##
[0049] The compounds of the general formula I have a number of
applications. They may for example be used for direct clinical
application to a patient, and accordingly, the present invention
further provides a pharmaceutical composition comprising a compound
of the general formula I together with a pharmaceutically
acceptable carrier. The invention further provides a compound of
the general formula I for use as a medicament, and a method of
treating a patient which comprises administering a
pharmaceutically-effective amount of a compound of the formula I or
a pharmaceutical composition according to the invention to the
patient. Any desired pharmaceutical effect, for example trauma
treatment, enzyme replacement, toxin removal, anti-inflammatory,
anti-infective, immunomodulatory, vaccination or anti-cancer, may
be obtained by suitable choice of biological molecule.
[0050] The compounds of the general formula I may also be used in
non-clinical applications. For example, many physiologically active
compounds such as enzymes are able to catalyse reactions in organic
solvents, and compounds of the general formula I may be used in
such applications. Further, compounds of the general formula I may
be used as diagnostic tools.
[0051] The following gives some specific biological molecules which
may have utility in the present invention, depending upon the
desired application. Enzymes include carbohydrate-specific enzymes,
proteolytic enzymes and the like. Enzymes of interest, for both
industrial (organic based reactions) and biological applications in
general and therapeutic applications in particular include the
oxidoreductases, transferases, hydrolases, lyases, isomerases and
ligases disclosed by U.S. Pat. No. 4,179,337. Specific enzymes of
interest include asparaginase, arginase, adenosine deaminase,
superoxide dismutase, catalase, chymotrypsin, lipase, uricase,
bilirubin osidase, glucose oxidase, glucuronidase, galactosidase,
glucocerbrosidase, glucuronidase, glutaminase
[0052] The biologically active molecules used in compounds of the
general formula I of the present invention include for example
factor 8, insulin, ACTH, glucagen, somatostatin, somatotropins,
thymosin, parathyroid hormone, pigmentary hormones, somatomedins,
erythropoietin, luteinizing hormone, hypothalamic releasing
factors, antidiuretic hormones, prolactin, interleukins,
interferons, colony stimulating factors, hemoglobin, cytokines,
antibodies, chorionicgonadotropin, follicle-stimulating hormone,
thyroid stimulating hormone and tissue plasminogen activator.
[0053] Certain of the above proteins such as the interleukins,
interferons and colony stimulating factors also exist in
non-glycosilated form, usually the result of preparation by
recombinant protein techniques. The non-glycosilated versions may
be used in the present invention.
[0054] For example, for Interferons, the invention permits the
preparation of conjugates in which the biological activity is
retained compared with non-conjugated Interferons. This most
surprising result is not possible using known conjugation
techniques.
[0055] Other proteins of interest are allergen proteins disclosed
by Dreborg et all Crit. Rev. Therap. Drug Carrier Syst. (1990) 6
315 365 as having reduced allergenicity when conjugated with a
polymer such as poly(alkylene oxide) and consequently are suitable
for use as tolerance inducers. Among the allergens disclosed are
Ragweed antigen E, honeybee venom, mite allergen and the like.
[0056] Glycopolypeptides such as immunoglobulins, ovalbumin,
lipase, glucocerebrosidase, lectins, tissue plasminogen activator
and glycosilated inerleukins, interferons and colony stimulating
factors are of interest, as are immunoglobulins such as IgG, IgE,
IgM, IgA, IgD and fragments thereof.
[0057] Of particular interest are antibodies and antibody fragments
which are used in clinical medicine for diagnostic and therapeutic
purposes. The antibody may used alone or may be covalently
conjugated ("loaded") with another atom or molecule such as a
radioisotope or a cytotoxic/antiinfective drug. Epitopes may be
used for vaccination to produce an immunogenic polymer-protein
conjugate.
[0058] A key feature of the process of the invention is that an
.alpha.-methylene leaving group and a double bond are
cross-conjugated with an electron withdrawing function that serves
as a Michael activating moiety. If the leaving group is prone to
elimination in the cross-functional reagent rather than to direct
displacement and the electron-withdrawing group is a suitable
activating moiety for the Michael reaction then sequential
intramolecular bis-alkylation can occur by consecutive Michael and
retro Michael reactions. The leaving moiety serves to mask a latent
conjugated double bond that is not exposed until after the first
alkylation has occurred and bis-alkylation results from sequential
and interactive Michael and retro-Michael reactions as described in
J. Am. Chem. Soc. 1979, 101, 3098-3110 and J. Am. Chem. Soc. 1988,
110, 5211-5212.). The electron withdrawing group and the leaving
group are optimally selected so bis-alkylation can occur by
sequential Michael and retro-Michael reactions.
[0059] It is also possible to prepare cross-functional alkylating
agents with additional multiple bonds conjugated to the double bond
or between the leaving group and the electron withdrawing group as
described in J. Am. Chem. Soc. 1988, 110, 5211-5212.
[0060] Since the cross-functionalised bis-alkylating reagents of
the type mentioned above undergo alkylation that is controlled by
Michael-retro Michael equilibria and since it is possible to
partially reduce a number from one to a greater than one disulfide
bonds in a protein in a controlled fashion significantly retaining
tertiary structure, it is then possible to have bis-alkylation
occur across the two sulfhydrals from cysteines of a given
disulfide bond. Such a sequence of reactions results in the
reannealling of the disulfide bridge with the bis-alkylating
reagent.
[0061] The thiol ether bonds formed upon conjugation to give a
compound of the formula I are, in general, hydrolytically stable in
aqueous solution. The reagents themselves are also hydrolytically
stable. In this context, a compound is regarded as being
hydrolytically stable if it does not undergo substantial
degradation at physiological pH and temperature up to 45.degree. C.
Degradation of less than 50% under these conditions over an eight
hour period is considered insubstantial.
[0062] It will be appreciated that this invention allows the
production of polymer reagents that possess cross-functionalised
bis-alkylating functionality at either the termini or on pendent
chains along the main chain of a polymer.
[0063] Some examples of novel conjugates according to the invention
include the following: ##STR15##
[0064] Some examples of novel reagents according to the invention
include the following: ##STR16##
[0065] The following Examples illustrate the invention. FIGS. 1 to
5 show the results obtained from Example 7.
EXAMPLE 1
Synthesis of Polymer Conjugating Reagent
p-Nitro-3-piperidinopropriophenone hydrochloride:
C.sub.14H.sub.19ClN.sub.2O.sub.3
[0066] To a single-neck 250 ml round-bottom flask was added
p-nitroacetophenone (16.5 g), paraformaldehyde (4.5 g), piperidine
hydrochloride (12.1 g), absolute ethanol (100 mL) and a magnetic
stir bar. To the stirred heterogeneous mixture was added
hydrochloric acid (37 wt % in water, 1 mL) and the solution was
heated to reflux under nitrogen. After a 1-2 h period more
paraformaldehyde (3.0 g) was added. The solution was allowed to
reflux for approximately 18 h during which time further
paraformaldehyde was added (3.0 g). After allowing the reaction
solution to cool a crystalline solid settled that would not
dissolve upon further refluxing. The solid was isolated by
filtration and recrystallised using very hot methanol to afford
large yellow crystals (10.9 g). .sup.1H NMR (DMSO-d.sub.6)
.delta.1.34-1.50 (m, 1H), 1.64-1.79 (m, 2H) , 1.79-1.94 (m, 4H),
2.89-3.05 (m, 2H), 3.41 (q, 2H), 3.51-3.54 (m, 2H), 3.82 (t, 2H),
8.26 (s, 1H), 8.29 (s, 1H), 8.41 (s, 1H), 8.44 (s, 1H).
3-(2-Hydroxyethylthiol)-p-nitropropiophenone
[0067] A stirred solution of p-nitro-3-piperidinopropiophenone
hydrochloride (30 g, 0.1 mol) and mercaptoethanol (9.5 g 0.12 mol)
in 95% ethanol (200 ml) was slowly heated until homogeneous.
Piperidine (1.0 ml) was added and the reaction mixture heated to
reflux for 2 h. After cooling the majority of the solvent was
rotoevaporated and ethyl acetate (200 ml) added and the solid
filtered. The ethyl acetate solution was extracted sequentially
with 10% aqueous HCl, 5% NaHCO.sub.3 and brine, and then dried over
Na.sub.2SO.sub.4, and then the ethyl acetate was evaporated to an
oil that crystallised to give 23.2 g of the desired product that
was recrystallised in ethyl acetate-ethyl ether.
2,2-Bis(p-tolylthiolmethyl)-p-nitroacetophenone:
C.sub.24H.sub.23NO.sub.3S.sub.2
[0068] To a 100 ml single-neck round-bottom flask was added
p-nitro-3-piperidinopropiophenone hydrochloride (10.0 g),
4-methylbenzenethiol (8.2 g), formaldehyde (37% w/w aq. solution,
10 ml, excess), methanol (40 ml) and a magnetic stir bar. The
stirred heterogeneous mixture was heated until a yellow homogeneous
solution formed (a couple of minutes at 50 - 60.degree. C.). Five
drops of piperidine were then added and the reaction solution
heated to reflux. Within 15 min the reaction became heterogeneous
due to the presence of some white/yellow solid and after 2 h this
solid became a strong orange in colour. The refluxing was stopped
after this time and the reaction was allowed to cool overnight to
room temperature. The reaction mixture was then heated under reflux
again with additional formaldehyde (37% w/w aq. solution, 10 ml,
excess). After refluxing for approximately 30 min an orange oil was
visible and no solid. The oil would settle to the bottom of the
flask when the stirring was stopped. After a further 7 h of
refluxing, the mixture was allowed to cool overnight, which
resulted in the settled oil crystallising. The crystalline solid
was isolated and purified by recrystallisation from very hot
methanol with several drops of acetone added to afford yellow
crystals (10.0 g). .sup.1H NMR (DMSO-d.sub.6) .delta.2.31 (s, 6H),
3.31-3.33 (m, 4H), 3.97 (quintet, 1H), 7.14 (q, 8H), 7.80 (d, 2H),
8.24 (d, 2H).
2, 2-Bis (p-tolylsulfonylmethyl) -p-nitroacetophenone:
C.sub.24H.sub.23NO.sub.7S.sub.2
[0069] In a 250 ml round-bottom flask a suspension of
2,2-bis(p-tolylthiolmethyl)-p-nitroacetophenone (2.5 g) and Oxone
(18.4 g) was stirred in a 1:1 methanol:water (100 ml) for 16 h.
This afforded a white solid suspension, to which was added
chloroform (100 ml) and the resulting organic phase was isolated
using a separating funnel to leave a white solid suspension within
the aqueous phase. Additional water was added to the aqueous phase
until a homogeneous solution formed, which was then washed again
with chloroform (100 ml). The organic phases were combined, washed
with brine (50 mL.times.2), dried with magnesium sulfate and the
solvent removed affording an off-white crude solid product after
drying in vacuo (2.5 g). The product was recrystallised from
acetone to give white crystals. .sup.1H NMR (CDCl.sub.3)
.delta.3.43-3.62 (m, 4H), 4.44 (quintet, 1H), 7.35 (d, 4H), 7.68
(d, 4H); 7.88 (d, 2H), 8.22 (d, 2H); analysis calculated for
C.sub.24H.sub.23NO.sub.7S.sub.2 (found): C, 57.47 (57.27); H, 4.62
(4.74); N 2.79 (2.58); MS (FAB) m/z 502 ([M+1].sup.+).
2-(2-Hydroxyethylsulfonylmethyl)-p-nitro-2(Z),
4-penta-dienophenone
[0070] To a flame dried round bottom flask purged with argon and
fitted with a thermometer and dropping funnel was added
3-(2-hydroxyethylthiol)-p-nitropropiophenone (0.5 g, 2.0 mmol) and
anhydrous tetrahydrofuran (50.0 ml). The solution was stirred and
cooled by a dry ice-acetone bath, then TiCl.sub.4 (0.23 ml, 2.1
mmol) was added by syringe. The ice bath was removed and the
solution allowed to warm to ambient temperature, then the solution
was cooled to -40.degree. C. and diisopropylethyl amine (1.1 ml)
was added by syringe. The cooling bath was removed and the reaction
mixture allowed to warm to -15 to 0.degree. C. turning a red
colour. The reaction mixture was then warmed to 25.degree. C. over
a 3-5 minute period and a solution of acrolein (0.14 g, 2.1 mmol)
in anhydrous tetrahydrofuran (20 ml) was added by dropping funnel
over a 30-40 minute period. The exothermic reaction caused the
temperature of the reaction mixture to increase to 30-40.degree. C.
and the solution stirred a further 20 minutes after addition of the
aldehyde before addition of ethyl acetate (75 ml). Thin layer
chromatography was used to confirm the disappearance of the
starting 3-(2-hydroxyethylthiol)-p-nitropropiophenone and the
formation of the desired 2-(2-hydroxyethylthiomethyl)-p-nitro-2(Z),
4 pentadienophenone (R.sub.f.about.0.38-0.45). The minor product
(E-isomer) could be observed at a lower R.sub.f in the range of
0.29-0.34. The ethyl acetate reaction mixture was extracted with
10% aqueous HCl and brine. The aqueous layers were combined and
extracted twice with ethyl acetate and all the ethyl acetate
fractions were combined and washed twice with 10% aqueous HCl, 5%
aqueous NaHCO.sub.3 and brine, then dried over solid
Na.sub.2SO.sub.4. The ethyl acetate was rotoevaporated to give the
crude 2-(2-hydroxyethylthiomethyl)-p-nitro-2(Z), 4
penta-dienophenone as an oil that was immediately oxidised as for
the synthesis of
2,2-bis[(p-tolylsulfonylmethyl)]-p-nitroacetophenone to give
2-(2-hydroxyethylsulfonylmethyl)-p-nitro-2(Z), 4-pentadienophenone
as a solid that was purified by column chromatography or
recrystallised in ethyl acetate or methanol and then covalently
bound to amino terminated poly(ethylene glycol) as described
elsewhere in this specification.
[0071] The above sequence of reactions was conducted with many
aldehydes including acealdehyde, methacrolein, ethacrolein
butryaldehyde, crotonaldehyde, 2,4-pentadienyal, sorb-aldehyde,
tolualdehyde, cinnamaldehyde, methyl-cinna-maldehyde,
chloro-cinnamaldehyde, 5-phenyl-2,4 pentadienal and
7-phenyl-2,4,6-heptatrienal. This sequence of reactions was
conducted with many aryl keto derivatives including
3-(p-tolylthiomethyl)-m-nitroacetophenone,
3-(2-hydroxyethylthio)-m-nitropropriophenone,
3-(ethylthiomethyl)-m-nitropropiophenone,
3-(dimethylaminoethylthio)-m-nitropropiophenone,
3-(2-hydroxyethylthio)-3-phenylpropriophenone,
3(2-hydroxyethylthiol)-5-phenyl-4(E)-pentenophenone,
3(ethylthiomethyl-o-nitropropiophenone,
3-(ethylthio)-propiophenone,
3-(2-hydroxyethylsulfonyl)propiophenone,
2-(3-(2-hydroxyethylthio)-1-propenyl)-m-2(E)-4-pentadienophenone.
This sequence of reactions was also conducted with aliphatic keto
derivatives including 4-(ethylthio)-2-butanone,
4-(-p-tolylthio)-2-butanone, 4-(4-nitrophenylthio)-2-butanone,
4-(2-hydroxyethylthio)-2-butanone and
methyl-3-(2-hydroxyethylthiol)-propanoate. The final products of
the aforementioned reactions for the these precursors can be
covalently bound to poly(ethylene glycol) as described elsewhere in
this specification.
2,2-Bis[(p-tolylsulfonyl)methyl]-p-aminoacetophenone hydrochloride
C.sub.24H.sub.26ClNO.sub.5S.sub.2
[0072] To a 100 mL round bottom flask was added
2,2-bis[(p-tolylsulfonyl)methyl]-p-nitroacetophenone (2 g), ethanol
(25 mL), hydrochloric acid (37 wt. % in water, 8 mL) and a magnetic
stir bar. To the resulting heterogeneous mixture was then added
tin(II) chloride dihydrate and the mixture heated in an oil bath at
45.degree. C. for 2 h. Water was then added to the homogeneous
yellow solution that had formed to a point where it appeared that a
precipitation may occur if more water was added. The homogenous
solution was allowed to cool to room temperature resulting in a
yellow compound crystallising/precipitating out, which was isolated
by filtration under vacuum. The isolated product was then mixed
with a heated mixture of acetone and methanol (approximately 90:10
v/v). An insoluble solid was isolated by filtration under vacuum
and dried to constant mass in a vacuum oven (1.4 g). .sup.1H NMR
(DMSO-d.sub.6) .delta.2.50 (s, 6H), 3.57-3.73 (overlapping m's,
5H), 6.27 (s, 2H), 6.39 (d, 2H), 6.96 (d, 2H), 7.47 (d, 4H), 7.55
(d, 4H).
Coupling of 2,2-bis[(p-tolylsulfonyl)methyl]-p-aminoacetophenone to
.alpha.-methoxy-.omega.-amino poly(ethylene glycol)
[0073] A one neck 100 mL round bottom flask fitted with a dropping
funnel and nitrogen line was charged with triphosgene (23 mg),
2,2-bis[(p-tolylsulfonyl)methyl]-p-aminoacetophenone hydrochloride
(125 mg), anhydrous toluene (2.5 mL) and a magnetic stir bar under
a nitrogen atmosphere. The dropping funnel was separately charged
with anhydrous triethylamine (68 .mu.L) and anhydrous toluene (2.5
mL). An acetone/dry-ice bath was placed below the round bottom
flask and the contents allowed to cool. The triethylamine solution
was then added dropwise to the triphosgene solution under stirring
over 5-10 min. The flask and dry-ice bath were allowed to warm to
room temperature which took several hours and once at room
temperature the reaction mixture was allowed to further stir for
about 2 h, still under a nitrogen atmosphere. A solution of
O-(2-aminoethyl)-O'-methylpoly(ethylene glycol) 2,000 (490 mg) and
anhydrous triethylamine (68 .mu.L) in anhydrous toluene was then
added dropwise to the reaction mixture at room temperature. The
resulting mixture was allowed to stir at room temperature overnight
(approximately 20 h total). The reaction mixture was then opened to
the atmosphere and filtered under gravity through a 5 mL disposable
syringe with a piece of non-absorbent cotton wool to act as a
filter. The homogeneous eluent was transferred to a 100 mL
separating funnel and then washed twice with deionised water (30 mL
and then 10 mL). The aqueous phases were combined and then washed
with diethyl ether (approximately 25 mL). The aqueous phase was
then freeze-dried to give an off-white solid product (160 mg).
Product was also found in diethyl ether and toluene phases. .sup.1H
NMR (CDCl.sub.3) .delta.2.5 (s), 3.39 (s), 3.41-3.53 (overlapping
m's), 3.53-3.76 (m), 3.82 (t), 4.17 (quintet), 7.35-7.39 (m, 1.39),
7.46 (d), 7.68 (d).
EXAMPLE 2
Synthesis of Polymer Conjugating Reagent
p-Carboxy-3-piperidinopropriophenone hydrochloride
[0074] To a 250 mL single-neck round-bottom flask was added
p-acetyl benzoic acid (10 g) and piperidine hydrochloride (7.4 g),
100 mL of absolute ethanol and a magnetic stir bar. To the stirred
heterogeneous mixture was added concentrated hydrochloric acid (1
mL) and the solution was then heated to reflux under nitrogen.
Paraformaldehyde (3.7 g) was added to the flask and refluxing
continued for approximately 1.5 h. A homogeneous solution formed to
which was added more paraformaldehyde (3.7 g). Heating was
continued for approximately 6 h during which time further
paraformaldehyde was added (3.7 g.). The reaction solution was
allowed to cool to room temperature and left for 1 week. A white
solid was isolated by filtration of the cooled reaction mixture. An
attempt was made to crystallise the solid after dissolving in very
hot methanol. An insoluble product (1.96 g) was isolated by
filtration and a second product (0.88 g) crystallised out of the
filtrate. Both products appeared identical by infra-red
spectroscopy and thin layer chromatography analysis after drying
in-vacuo and so were combined to be used in subsequent reactions.
ATR-FT-IR 1704, 1691, 1235, 760.
4-[2,2-Bis[(p-tolylthio)methyl]acetyl]benzoic acid:
C.sub.25H.sub.24O.sub.3S.sub.2
[0075] To a 50 mL single-neck round-bottom flask was added
p-carboxy-3-piperidinopropriophenone hydrochloride (2.5 g),
4-methylbenzenethiol (2.1 g) formaldehyde (37% w/w aq. solution,
2.5 mL), ethanol (10 mL), a magnetic stir bar and piperidine
(approximately ten drops). A condenser was then fitted to the flask
and the reaction solution heated to reflux. Methanol (5 mL) was
then added. After about 2 h, additional formaldehyde (2.5 mL) was
added and the heating continued for a further 2 h. The reaction
flask was then allowed to cool to room temperature whereupon the
reaction solution was diluted with diethyl ether (approximately 150
mL). The resulting organic phase was then washed with water
(acidified using 1N hydrochloric acid to pH 2-3; 50 mL.times.2),
water (50 mL) and brine (75 mL) and then dried over magnesium
sulfate. Filtration followed by removal of volatiles on a rotary
evaporator afforded a solid residue. The solid was dissolved in a
minimum volume of a mixture of predominately methanol and acetone
with heating. The homogeneous solution was then placed in a freezer
overnight, affording off-white crystals, which were isolated by
filtration under vacuum, washed with fresh acetone and then dried
to constant mass in a vacuum oven (2.5 g): .sup.1H NMR (CDCl.sub.3)
.delta.2.38 (s, 6H) , 3.16-3.31 (m, 4H), 3.85 (quintet, 1H), 7.15
(d, 4H), 7.18 (d, 4H), 7.64 (d, 2H), 8.07 (d, 2H); analysis
calculated for C.sub.25H.sub.24O.sub.3S.sub.2 (found): C, 68.78
(68.84); H, 5.54 (5.77).
4-[2,2-Bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid
C.sub.25H.sub.24O.sub.7S.sub.2
[0076] In a 250 mL round-bottom flask a suspension of
4-[2,2-bis[(p-tolylthio)methyl]acetyl]benzoic acid (2 g) and Oxone
(16.9 g) was stirred in 1:1 v/v methanol:water (100 mL) for 16 h.
This afforded a white solid suspension to which was added
chloroform (100 mL) and the resulting organic phase was isolated
using a separating funnel to leave a white solid suspension with
the aqueous phase. To the heterogenous queous phase was added
additional water until homogenous (approximately 170 mL) and then
the aqueous phase was washed with chloroform (75 mL). The organic
phases were combined, washed with water (50 mL.times.2, acidified
with a few drops of 1N hydrochloric acid) and brine (50 mL). The
organic phase was dried with magnesium sulfate, filtered and the
solvent removed using a rotary evaporator to give an off-white
crude solid product (2.2 g). Further purification (on 1 g of
product) was performed by recrystallisation from very hot ethyl
acetate, acetone and hexane affording 0.6 g of product: .sup.1H NMR
(CDCl.sub.3) .delta.2.51 (s, 6H), 3.49-3.72 (m, 4H), 4.44 (quintet,
1H), 7.40 (d, 4H), 7.73-7.78 (m, 6H), 8.13 (d, 2H); analysis
calculated for C.sub.25H.sub.24O.sub.7S.sub.2 (found): C, 59.98
(59.88); H, 4.83 (4.78).
Coupling of 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid
to .alpha.-methoxy-.omega.-amino PEG (2000 g/mol)
[0077] A one-neck 50 mL schlenk flask was charged with
4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (100 mg)
and a magnetic stir bar. The flask was sealed and a strong vacuum
applied for approximately 15 min. An argon atmosphere was
introduced into the flask and thionyl chloride (1 mL) added by
syringe with stirring. The resulting mixture was heated at
50.degree. C. for 2 h. Volatile liquids were then removed in vacuum
to afford a yellow foam. An argon atmosphere was again introduced
to the flask and anhydrous dichloromethane (5 mL) was added by
syringe to afford a homogeneous solution. Volatile liquids were
then removed again in vacuum. The solvent addition/removal process
was repeated until a white residue remained. Anhydrous
dichloromethane (5 mL) was again added to the schlenk flask to form
a homogeneous solution. Separately in a 25 mL round bottom flask
fitted with a septum and with a magnetic stir bar,
O-(2-aminoethyl)-O'-methylpoly(ethylene glycol) (2,000 g/mol) (0.2
g), and anhydrous triethylamine (30 .mu.L) were dissolved in
anhydrous dichloromethane (5 mL) under an argon atmosphere. The
reaction solution with bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic
acid was injected into the flask containing the poly(ethylene
glycol) solution in a dropwise fashion, immediately resulting in
the evolution of a white gas. The resulting solution was allowed to
stir overnight at room temperature whereupon additional
triethylamine (28 .mu.L, was added). After a further 1 h, the
reaction solution was added dropwise through a glass pipette into
rapidly stirred diethyl ether. To achieve precipitation it was
necessary to add hexane to the diethyl ether solution and place the
flask in an ice bath. The precipitant obtained was isolated by
centrifugation and dried in a vacuum oven to constant weight
affording an off-white solid product (230 mg). Further purification
was achieved by precipitation by first dissolving the product in
dichloromethane and adding to a chilled stirred diethyl ether
solution. .sup.1H NMR (CDCl.sub.3) .delta.2.51 (s), 3.40 (s),
3.60-3.75 (m), 3.84 (t), 4.36 (quintet), 7.39 (d), 7.68 (d), 7.71
(d), 7.85 (d).
Coupling of 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid
to .alpha.-methoxy-.omega.-amino PEG (20,000 g/mol)
[0078] A one-neck 50 mL Schlenk flask was charged with
4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (100 mg)
and a magnetic stir bar. The flask was sealed and a strong vacuum
applied for approximately 15 min. An argon atmosphere was
introduced into the flask and thionyl chloride (1 mL) added by
syringe with stirring. The resulting mixture was heated at
50.degree. C. for 2 h. After allowing the flask to cool to room
temperature, volatile liquids were removed in vacuum to afford a
yellow foam. An argon atmosphere was again introduced to the flask
and anhydrous dichloromethane (5 mL) was added by syringe to afford
a homogeneous solution. Volatile liquids were then removed again in
vacuum. The solvent addition-removal process was repeated until a
white residue remained. Finally, anhydrous dichloromethane (5 mL)
was added to the flask under argon to form a homogeneous
solution.
[0079] In a separate 50 mL Schlenk flask,
O-(2-aminoethyl)-O'-methylpoly(ethylene glycol) (20,000g/mol) (0.5
g) was dissolved in 4 mL of anhydrous toluene in an argon
atmosphere and then the solution was evaporated to dryness in
vacuum. The white residue afforded was kept in vacuum for 3 h and
then dissolved in anhydrous dichloromethane (5 mL) in an argon
atmosphere.
[0080] To the poly(ethylene glycol) solution, which was stirred and
cooled in an ice-water bath, was slowly added the reaction solution
of bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid drop-wise.
After complete addition 40 .mu.L of dried triethylamine was slowly
added to the resulting solution drop-wise. When the triethylamine
had been added the resulting solution was allowed to stir overnight
and warm to room temperature.
[0081] The reaction solution was then filtered through a 0.45 .mu.m
filter and volatile liquids removed from the eluent to afford a
slightly yellow/orange residue that was redissolved in warm acetone
(10 mL). The flask containing the resulting homogeneous solution
was placed in an ice-water bath and under stirring a precipitation
occurred. The white precipitate was isolated on a no. 3 sintered
glass funnel and washed with chilled fresh acetone (approximately
30 mL). The isolated solid was allowed to dry in vacuum to afford
0.4 g of white solid. .sup.1H NMR (CDCl.sub.3) .delta.2.42 (s),
3.31 (s), 3.40-3.75 (m), 4.27 (m), 7.30 (d), 7.59 (d), 7.63 (d),
7.75 (d).
[0082] The
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzam-
ide poly(ethylene glycol) conjugating reagent (20,000 g/mol) (0.4
g) was dissolved in 10 mL of an argon purged mixture of
acetonitrile:water (24:1 v/v, 2 mg hydroquinone) and the solution
filtered through a 0.45 .mu.m PP filter. The eluent was placed
under argon in a round-bottom flask and triethylamine (50 .mu.L)
was added under stirring. The flask was then placed in a 30.degree.
C. oil bath for 20 h with stirring after which time the flask was
cooled to room temperature and the volatile liquids removed in
vacuum. The residue obtained was dissolved in warm acetone (10 mL)
and once homogeneous the solution was placed in an ice-water bath
whereupon precipitation occurred while the solution stirred. The
white precipitate was isolated on a no. 3 sintered glass funnel and
washed with chilled fresh acetone (approximately 30 mL). The
isolated solid was allowed to dry under vacuum to afford 0.3 g of
white solid. .sup.1H NMR (CDCl.sub.3) .delta.2.35 (s), 3.31 (s),
3.39-3.78 (m), 4.28 (s), 5.84 (s), 6.22 (s), 7.26 (d), 7.65 (d),
7.72 (d), 7.81 (d)
EXAMPLE 3
Reaction of 4-methylbenzenethiol and
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzami-
de poly(ethylene glycol)
[0083] The polymer conjugating reagent,
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzami-
de poly(ethylene glycol) (2000 g/mol) (30 mg, 12.1 .mu.mol, 1 eq.)
and 4-methylbenzenethiol (3 mg, 24.2 mmol, 2 eq.) were dissolved in
deuterated chloroform (approximately 0.75 mL). To the homogeneous
solution was then added triethylamine (1.7 .mu.L, 12.1 .mu.mol, 1
eq.). The reaction mixture was stirred and a H-NMR spectrum was
obtained. The resulting spectrum confirmed that addition of the
4-methylbenzenethiol to the polymer conjugating reagent had
occurred. A similar reaction was conducted with propane triol.
EXAMPLE 4
Polymer Conjugation to Ribonuclease A
[0084] To Ribonuclease A (30 mg) in a 15 mL centrifuge tube was
added 3 mL of an 8M aqueous urea solution, followed by
2-mercaptoethanol (60 .mu.L). The pH of the resulting solution was
adjusted to pH 8.5 using a 10% aqueous solution of methylamine. The
reaction solution was then bubbled with nitrogen for approximately
30 min. Still purging with nitrogen the tube was heated at
37.degree. C. for 5 h. The reaction mixture was then cooled in an
ice-salt water bath and 10 mL of an argon purged chilled solution
of 1N HCl:absolute ethanol (1:39 v/v) was added to the reaction
solution. A precipitation occurred and the precipitate was isolated
by centrifugation and then washed three times with further 10 mL
portions of the HCl:absolute ethanol mixture and twice with
nitrogen purged chilled diethyl ether (2.times.10 mL). After each
washing the precipitate was isolated by centrifugation. The washed
precipitate was then dissolved in nitrogen purged de-ionised water
and freeze-dried to afford a dry solid. Partial reduction of
Ribonuclease A was confirmed and quantitated using Ellman's Test,
which gave 5.9 free thiols per protein molecule.
[0085] In an eppendorf, the partially reduced Ribonuclease A (10.9
mg) was dissolved in argon purged pH 8 ammonia solution (500
.mu.L). In a separate eppendorf, the polymer conjugating reagent,
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzam-
ide derived from poly(ethylene)glycol (2000 g/mol) (5 mg) was also
dissolved in ammonia solution (250 .mu.L) and the resulting
solution added to the Ribonuclease A solution. The PEG eppendorf
was washed with 250 .mu.L of fresh ammonia solution and this was
also added to the main reaction eppendorf. The reaction eppendorf
was then closed under argon and heated at 37.degree. C. for
approximately 24 h and then allowed to cool to room temperature.
The cooled reaction solution was then analysed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE
experiment was consistent with reaction of pegylated
4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]-benzoic acid with
Ribonuclease A.
EXAMPLE 5
Polymer Conjugation to Mouse IgG Antibody Fab Fragment
[0086] To 240 .mu.L of a 0.4 mg/mL solution of mouse IgG antibody
Fab fragment (Abcam Cat. No. AB6668) in an argon purged 0.02 M
sodium phosphate pH 6 buffer containing 0.15 M sodium chloride and
0.005 M EDTA was added 4 .mu.L of an argon purged aqueous solution
of 1 mM selenocystamine dihydrochloride, followed by 12 .mu.L of an
argon purged aqueous solution of tris(2-carboxyethyl) phosphine
hydrochloride (TCEP) at room temperature. The resulting solution
was immediately vortexed for several seconds and then allowed to
stand at room temperature for 6 min. Two 5 .mu.L samples of the
solution containing reduced Fab fragment were removed for future
analysis and the remaining solution was then allowed to stand in an
ice-water bath for 4 min. An ice-water bath chilled solution of
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzami-
de derived from poly(ethylene glycol) (20,000 g/mol) (1.6 .mu.L, 50
mg/ml, argon purged 0.02 M sodium phosphate pH 6 buffer containing
0.15 M sodium chloride, 0.005 M EDTA and 0.23 mM hydroquinone) was
immediately added to the Fab solution; the solution vortexed for
several seconds and then placed back into the ice-water bath. A
further 1.6 .mu.L of the conjugation reagent solution was added in
an analogous manner every two minutes until 6.4 .mu.L had been
added in total. After complete addition the reaction solution was
placed in a refrigerator (<5.degree. C.) for 2 h. A sample of
the reaction solution was then removed for analysis by SDS
PAGE.
[0087] The reaction mixture was analysed by SEC-HPLC (100 .mu.l
injection, superpose 12-24 ml column, Amersham biosciences; eluent:
20 mM Na Phosphate, 0.15 M NaCl pH 7.0; isocratic elution for 100
min at a flow of 0.25 ml/min; UV detection, 210 nm) displaying the
Fab-poly(ethylene glycol) conjugate that was not present in the
identical control reaction mixture. Fractions isolated from
SEC-HPLC chromatography were analysed by SDS PAGE and the mono
poly(ethylene glycol) conjugate was observed.
EXAMPLE 6
Polymer Conjugation to Asparaginase
[0088] A 100 .mu.l sample of a 5 mg/ml solution of asparaginase at
pH 6.5 (Sigma) was diluted with 900 .mu.l of 20 mM phosphate buffer
(pH 6 and also containing 0.15 M NaCl and 5 mM EDTA).
DL-dithioreitol (DTT, 15.4 mg) was then added and the resulting
solution allowed to stand at room temperature. After allowing to
stand for 2 h the solution was purified on a PD-10 column
(Sephadex.RTM.G-25M, Pharmacia Biotech) that had been equilibrated
with 20 mM phosphate buffer (pH 6 and also containing 0.15 M NaCl
and 5 mM EDTA). The column was eluted with 1 ml portions of fresh
buffer. Two fractions containing reduced protein were identified
using UV spectroscopy at 280 nm. These fractions were concentrated
using centrifugal filter devices (MWCO 3,000; Microcon) to a volume
of approximately 270 .mu.l and then diluted to 1 ml with fresh pH 6
phosphate buffer (additionally containing 0.23 mM hydroquinone).
Two 5 .mu.l samples were removed for later analysis by SDS PAGE.
Separately, a 50 mg/ml solution of the conjugating reagent solution
of
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzami-
de derived from poly(ethylene glycol) (20,000 g/mol) was made up in
the same phosphate buffer and placed in an ice-water bath along
with the protein solution for 5 min. To the protein solution was
then added 58 .mu.l of the conjugation reagent solution and after
several seconds of vortexing, the reaction solution was placed in a
refrigerator (<5.degree. C.). A control reaction was also
performed under the same conditions and scale but using non-reduced
asparaginase. Successful poly(ethylene glycol) conjugation to
asparaginase was confirmed by SDS PAGE (4-12% Bis-Tris Gel with
colloidal blue staining) performed with samples taken from the
reaction solution after 2 h. No conjugation band was observed by
SDS PAGE for a sample taken from a control reaction where the
asparaginase was not allowed to react with DTT.
EXAMPLE 7
Polymer Conjugation to Interferon
[0089] A solution of 100 .mu.l interferon .alpha.-2b (Shantha
Biotechnics) (1 mg/ml) diluted with 150 .mu.l of a buffer solution
(sodium phosphate 0.02 M; NaCl 0.15 M; EDTA 0.005 M; pH 6.0 in
argon purged deionised water) was partially reduced by the addition
of selenocystamine (1 mM in argon purged deionised water, 2
equivalents) and TCEP (1 mM in argon purged deionised water, 5
equivalents). The protein solution was cooled to 4.degree. C. The
conjugation reagent
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzami-
de derived from poly(ethylene glycol)(20,000 g/mol) was dissolved
in the buffer solution (50 mg/ml), cooled to 4.degree. C. and 16
.mu.l added to the protein solution. The reaction mixture was
vortexed and incubated at 4.degree. C. for 2 h. The non-ionic
components of the reaction mixture were removed by cation exchange
chromatography (Hitrap SP FF 1 ml column, Amersham biosciences)
using two buffers: (A) 25 mM Na Acetate pH 4.0 and (B) 25 mM Na
Acetate +0.7 M NaCl pH 4.0. The column was washed (1 ml/min) with
buffer A (5.0 ml), then buffer B (5.0 ml) and then equilibrated
with 10 ml of buffer A (10 ml). The conjugate was loaded on the
column (200 .mu.l) followed by buffer A (500 .mu.l) and a loading
fraction (0.5 ml) was collected. The column was washed with 5 ml of
buffer A and fractions were collected. The sample was eluted from
the column with buffer B (10 ml) and fractions were collected.
Spectroscopy (UV, 280 nm) was used to determine the fractions that
contained protein and these fractions were then purified into
separate fractions by size exclusion chromatography (100 .mu.l
injections, superose 12-24 ml column, Amersham biosciences; eluent:
20 mM Na Phosphate, 0.15 M NaCl pH 7.0; isocratic elution for 100
min at a flow of 0.25 ml/min; UV detection, 210 nm). These
purification conditions provided base line resolution for the
separation of the poly(ethylene glycol) interferon conjugate from
native interferon. The conjugation reaction and purification of the
poly(ethylene glycol) conjugate were confirmed by SDS-PAGE; 12%
Bis-Tris gel (SilverQuest Silver Staining; Colloidal Blue Staining
and perchloric acid 0.1 M/BaCl.sub.2 5% and I.sub.2 0.1 M
staining). No conjugation was observed when a solution of 40 .mu.l
of interferon (1 mg/ml) in 60 .mu.l of sodium phosphate 0.02 M,
NaCl 0.15 M, EDTA 0.005 M, pH 6.0 in argon purged water was mixed
with the conjugation reagent
.alpha.-methoxy-.omega.-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzami-
de derived from poly(ethylene glycol) (20,000 g/mol) was dissolved
in the buffer solution (50 mg/ml), cooled to 4.degree. C. and 16
.mu.l added to the protein solution. This reaction mixture was
vortexed and incubated at 4.degree. C. for 2 h.
[0090] The concentration of each purified fraction of the
poly(ethylene glycol) interferon conjugate was determined by enzyme
immuno assay. The same assay was utilised to determine the
concentration of the native interferon that was conjugated and an
international standard NIBSC (UK) sample of interferon. The
reproducible and accurate standard curve obtained for all forms of
the aforementioned interferon is shown in FIG. 1. A549 (human lung
fibroblast) cells were plated at 15,000 cells/well in 96-well
flat-bottom tissue culture plates. Interferon from the NIBSC (UK),
the native interferon that was conjugated and the poly(ethylene
glycol) interferon conjugate were then separately added to the
cells in triplicate and the plate incubated at 37.degree. C. (5%
CO.sub.2) for 24 h. On the next day, a working solution of EMCV
(encephalomyocarditis virus) was prepared in DMEM/2% FCS from stock
virus stored at -80.degree. C. The media containing the interferon
or the poly(ethylene glycol) interferon conjugate was removed and
replaced with media containing EMCV. The tissue culture plate was
then incubated for 1 h at, 37.degree. C. after which the virus was
removed. One hundred microlitres of DMEM/10% FCS media was added
and the plate incubated for 16-24 h at 27.degree. C. From 16 h
onwards, the plate was read regularly to ascertain when cell death
was starting. The media was then aspirated and the cells washed
with 100 .mu.l of phosphate buffer solution. This was followed by
the addition of 50 .mu.l of methyl violet solution [i.e., methyl
violet (4% formaldehyde, 0.05% methyl violet 2B (Sigma-Aldrich)]
for 30 min at room temperature. The plate was then washed with 100
.mu.l of water and dried in air. Spectrophotometric absorbance was
determined at 570 nm using a plate reader. FIG. 2 confirms that the
interferon used for the conjugation had equivalent activity of the
international standard NIBSC (UK) interferon. FIG. 3 confirms that
native interferon used for conjugation maintained equivalent
biological activity after being subjected to all the chemical and
purification processes except for exposure with the poly(ethylene
glycol) conjugation reagent. FIG. 4 confirms that the native
interferon used for conjugation and the purified poly(ethylene
glycol) interferon conjugate maintained equivalent biological
activity.
[0091] Induction of 2'5'-oligoadenylate synthetase (2,5'-OAS) and
protein kinase R (PRKR) mRNA by interferon-.alpha. 2b was
determined. The purified poly (ethylene glycol) interferon
conjugate and a sample of the native interferon-.alpha. 2b were
evaluated. Two million MOLT 4 cells/well were incubated in 24 well
tissue culture plates with 5000 pg of each interferon sample as
measured using an enzyme immunoassay for 24 h at 37.degree.. The
total RNA was extracted (RNA II Isolation kit, Macheray-Nagel) and
200 ng subjected to reverse transcription in a final volume of 20
.mu.L (Sigma, AMV reverse transcription kit). Samples were diluted
1 in 4 in water and 2 .mu.L of each sample amplified in a 20 .mu.L
real-time PCR quantitation mix (Sigma SybrGreen ReadyMix). The
primers used were: TABLE-US-00001 2'5'-OAS forward GGC TAT AAA CCT
AAC CCC CAA ATC 2'5'-OAS reverse AGC TTC CCA AGC TTC TTC TTA CAA
PKR forward ACT CTT TAG TGA CCA GCA CAC TCG PRKR reverse TTT AAA
ATC CAT GCC AAA CCT CTT
[0092] For 2',5'-OAS amplification, enzyme activation at 94.degree.
C. for 5 min was followed by 48 cycles of denaturation at
94.degree. C. for 5 sec, annealing at 60.degree. C. for 2 sec and
extension at 72.degree. C. for 8 sec. For PRKR amplification,
samples were activated at 94.degree. C. for 5 min and underwent 48
cycles of denaturation at 94.degree. C. for 5 sec, annealing at
59.degree. C. for 2 sec and extension at 72.degree. C. for 8 sec. A
melting curve analysis of the products was performed at the end of
the amplifications. Quantitation of induced mRNA levels was done
relative to the control untreated cells using the following
formula: Relative increase=2.sup.-(Ct sample-Ct control) where Ct
is the threshold cross-over value.
[0093] FIG. 5, in which FIG. 5(a) shows the real-time quantitative
RT-PCR (two-step) analysis of interferon-inducible
2'5'-Oligoadenylate Synthetase (2'5'-OAS)gene, and FIG. 5(b) shows
the real-time quantitative RT-PCR (two-step) analysis of
interferon-inducible Protein Kinase R (PRKR) gene. The results
confirm that the poly(ethylene glycol) interferon-.alpha.2b
conjugate stimulated 2'5'-oligoadenylate synthetase (2'5'-OAS and
protein kinase R (PRKR) mRNA synthesis to levels that were similar
to native unpegylated interferon-.alpha. 2b.
* * * * *